Dentin Materials as Biological Scaffolds for Tissue Engineering

Vital tooth-derived demineralized dentin matrix (DDM) has a bone-inductive ability, while non-vital tooth-derived DDM lost it. Acid treatment for dentin provides the increase of surface area, the release of matrix-binding growth factors such as BMPs, and the decrease of the infection risk. Human autograft of vital tooth-derived DDM was achieved first in Japan 2002, while first bone autograft was noted in Italy 1820. This paper introduced dentin/bone biology and a unique clinical case, combined with two types of non-vital tooth-derived DDM (roots, granules) for lateral bone augmentation. A 63-year-old woman revealed highly atrophic mandible in 2015. Three non-vital teeth were extracted, changed in shape, demineralized in 2% HNO3, were rinsed, and were grafted immediately. The CT images at 3 months after the graft showed remarkable lateral augmentation. DDM scaffolds were received to host, and two fixtures were placed into the DDM-augmented bone. The patient was successfully restored with their own DDM scaffolds and implant surgery

Chemical Properties of Human Dentin Blocks and Vertical Augmentation by Ultrasonically Demineralized Dentin Matrix Blocks on Scratched Skull without Periosteum of Adult-Aged Rats

Vertical augmentation is one of the most challenging techniques in bone engineering. Several parameters, such mechano-chemical characteristics, are important to optimize vertical bone regeneration using biomaterials. The aims of this study were to chemically characterize human dentin blocks (calcified demineralized dentin matrix: CDM, partially demineralized dentin matrix: PDDM and completely demineralized dentin matrix: CDDM) (2 × 2 × 1 mm3) chemically and evaluate the behavior of PDDM blocks on non-scratched or scratched skulls without periosteum of adult rats (10–12 months old, female) as a vertical augmentation model. The dissolved efficiency of CDM showed 32.3% after ultrasonic demineralization in 1.0 L of 2% HNO3 for 30 min. The 30 min-demineralized dentin was named PDDM. The SEM images of PDDM showed the opening of dentinal tubes, nano-microcracks and the smooth surface. In the collagenase digestion test, the weight-decreasing rates of CDM, PDDM and CDDM were 9.2%, 25.5% and 78.3% at 12 weeks, respectively. CDM inhibited the collagenase digestion, compared with PDDM and CDDM. In the PDDM onlay graft on an ultrasonically scratched skull, the bone marrow-space opening from original bone was found in the bony bridge formation between the human PDDM block and dense skull of adult senior rats at 4 and 8 weeks. On the other hand, in the cases of the marrow-space closing in both non-scratched skulls and scratched skulls, the bony bridge was not formed. The results indicated that the ultrasonic scratching into the compact parietal bone might contribute greatly to the marrow-space opening from skull and the supply of marrow cells, and then bony bridge formation could occur in the vertical augmentation model without a periosteum

Accelerated Bone Induction of Adult Rat Compact Bone Plate Scratched by Ultrasonic Scaler Using Acidic Electrolyzed Water

Fresh compact bone, the candidate graft material for bone regeneration, is usually grafted for horizontal bone augmentation. However, the dense calcified structure inhibits the release of growth factors and limits cellular and vascular perfusion. We aimed to create mechano-chemically altered dense skull bone by ultrasonic treatment, along with partial demineralization using commercially available acidic electrolyzed water (AEW). The parietal skull bone of an 11-month-old Wistar rat was exposed and continuously treated with a piezoelectric ultrasonic scaler tip for 1 min, using AEW (pH 2.3) or distilled water (DW, pH 5.6) as irrigants. Treated parietal bone was removed, cut into plates (5 × 5 × 1 mm3), grafted into the back subcutaneous tissues of syngeneic rats, and explanted at 1, 2, and 3 weeks. AEW bone showed an irregular surface, deep nano-microcracks, and decalcified areas. SEM-EDS revealed small amounts of residual calcium content in the AEW bone (0.03%) compared to the DW bone (0.86%). In the animal assay, the AEW bone induced bone at 2 weeks. Histomorphometric analysis showed that the area of new bone in the AEW bone at 2 and 3 weeks was significantly larger. This new combination technique of AEW-demineralization with ultrasonic treatment will improve the surface area and three-dimensional (3D) architecture of dense bone and accelerate new bone synthesis

Human Fresh Fibrin Membrane with Bone Morphogenetic Protein-2 (BMP-2) Induces Bone Formation in the Subcutaneous Tissues of Nude Mice

Autologous blood-derived fibrin glue with platelets, called the concentrated growth factor (CGF), can be prepared immediately by only the decided centrifuge without the addition of coagulation factors. Collagen materials combined with recombinant human BMP-2 have been commercially available for clinical use. The fresh CGF is auto-clot with wettability and elasticity, while most collagen membranes are derived from the cow or pig. The fresh CGF has wettability and elasticity, while collagen membranes are dry materials without elasticity. The aim of this study was to observe the microstructures of human CGF membrane and evaluate its behavior as a delivery scaffold of rhBMP-2 in the subcutaneous tissues of nude mice. Twenty-four nude mice (5-week-old, male) were used for the assessment of in vivo ectopic bone formation. Mice were received the CGF membrane as the controls and the CGF/rhBMP-2 membrane as the experimental group in the subcutaneous tissues, and harvested at 7, 10, and 14 days after the graft. Harvested samples were evaluated for the histological examination and the histomorphometric measurement was conducted to compare the residue of the CGF, as well as the new bone. Mature fibrin fibers assembled from multiple fibrillary elements and platelets with the rhBMP-2 membrane induced several bony islands and cartilage without residues of CGF at 14 days, while the CGF membrane alone was almost absorbed at 10 days and failed to induce bone formation at 14 days. These results demonstrated that the fresh, human CGF membrane could contribute to a short-term, sticky fibrin matrix for the delivery of rhBMP-2

Mechanical Properties of Human Concentrated Growth Factor (CGF) Membrane and the CGF Graft with Bone Morphogenetic Protein-2 (BMP-2) onto Periosteum of the Skull of Nude Mice

Concentrated growth factor (CGF) is 100% blood-derived, cross-linked fibrin glue with platelets and growth factors. Human CGF clot is transformed into membrane by a compression device, which has been widely used clinically. However, the mechanical properties of the CGF membranes have not been well characterized. The aims of this study were to measure the tensile strength of human CGF membrane and observe its behavior as a scaffold of BMP-2 in ectopic site over the skull. The tensile test of the full length was performed at the speed of 2mm/min. The CGF membrane (5 × 5 × 2 mm3) or the CGF/BMP-2 (1.0 μg) membrane was grafted onto the skull periosteum of nude mice (5-week-old, male), and harvested at 14 days after the graft. The appearance and size of the CGF membranes were almost same for 7 days by soaking at 4 °C in saline. The average values of the tensile strength at 0 day and 7 days were 0.24 MPa and 0.26 MPa, respectively. No significant differences of both the tensile strength and the elastic modulus were found among 0, 1, 3, and 7 days. Supra-periosteal bone induction was found at 14 days in the CGF/BMP-2, while the CGF alone did not induce bone. These results demonstrated that human CGF membrane could become a short-term, sticky fibrin scaffold for BMP-2, and might be preserved as auto-membranes for wound protection after the surgery

Selective ablation of basophils in mice reveals their nonredundant role in acquired immunity against ticks

Ticks are ectoparasitic arthropods that can transmit a variety of microorganisms to humans and animals during blood feeding, causing serious infectious disorders, including Lyme disease. Acaricides are pharmacologic agents that kill ticks. The emergence of acaricide-resistant ticks calls for alternative control strategies for ticks and tick-borne diseases. Many animals develop resistance to ticks after repeated infestations, but the nature of this acquired anti-tick immunity remains poorly understood. Here we investigated the cellular and molecular mechanisms underlying acquired resistance to Haemaphysalis longicornis ticks in mice and found that antibodies were required, as was IgFc receptor expression on basophils but not on mast cells. The infiltration of basophils at tick-feeding sites occurred during the second, but not the first, tick infestation. To assess the requirement for basophil infiltration to acquired tick resistance, mice expressing the human diphtheria toxin receptor under the control of the mast cell protease 8 (Mcpt8) promoter were generated. Diphtheria toxin administration to these mice selectively ablated basophils. Diphtheria toxin–mediated basophil depletion before the second tick infestation resulted in loss of acquired tick resistance. These data provide the first clear evidence, to our knowledge, that basophils play an essential and nonredundant role in antibody-mediated acquired immunity against ticks, which may suggest new strategies for controlling tick-borne diseases